Inhibition Of MAPK Pathway Research Articles

C-C Motif Chemokine 2 Regulates Macrophage Polarization and Contributes to Myocardial Infarction Healing.

Macrophages are crucial immune cells that play essential roles in the healing of myocardial infarction (MI), undergoing continuous polarization throughout this process. C-C motif chemokine 2 (CCL2) is a chemokine that regulates inflammatory responses during MI. However, the extent to which CCL2 influences macrophage polarization and MI healing remains incompletely understood. In this study, we investigate the role of CCL2 in macrophage polarization and MI healing. Our findings reveal that CCL2 is differentially expressed in lipopolysaccharide (LPS)-induced M1 and interleukin (IL)-4-induced M2 RAW264.7 macrophages. Knockdown of CCL2 attenuates TNF-α secretion stimulated by LPS, while overexpression of CCL2 mitigates IL-10 production triggered by IL-4 in these macrophages. Moreover, CCL2 deficiency disrupts LPS-induced M1 polarization, whereas CCL2 overexpression reduces M2 polarization of RAW264.7 macrophages induced by IL-4. Further exploration indicates that the promotion of M1 polarization by CCL2 is significantly impaired by inhibition of the p38-mediated MAPK pathway and NF-κB pathway. In a MI mouse model, CCL2 knockdown remarkably reduces infarct size, collagen synthesis, and the expression of cardiac fibrosis and hypertrophy markers. The activity of the p38-mediated MAPK pathway and NF-κB pathway is downregulated by CCL2 knockdown as well. Additionally, the number of total macrophages and M1 macrophages in the infarct decreases, while the number of M2 macrophages increases upon CCL2 deficiency. In conclusion, these results suggest that CCL2 is a key regulator of macrophage polarization, controlling MI healing in vivo.

Bacteroides thetaiotaomicron and its inactivated bacteria ameliorate colitis by inhibiting macrophage activation

BackgroundStudies have demonstrated that Bacteroides thetaiotaomicron (BT) has protective effect against colon inflammation in murine models. Macrophages play an important role in gut immunity. However, the specific mechanisms of BT on macrophage are still unelucidated. Thus, our study investigates the anti-inflammatory effect of BT and its heat-treated inactivated bacteria on experimental colitis and macrophages. MethodsA dextran sulfate sodium (DSS)-induced acute colitis model with male C57BL/6 mice, BT (ATCC29148) strain, THP1 cell lines were used in this study. Live and heat-treated inactivated BT (IBT) solution (1 × 10^9cfu/ml) were intragastrically gavaged daily for 14 days. Colonic inflammation was determined by the disease activity index (DAI) score, colon length, histological score, and inflammatory factors. THP1 cells were induced towards M1, then treated with different concentrations of inactivated BT solution and p38 inhibitor. Western blotting, immunohistochemistry, immunofluorescence and qRT-PCR were performed to assess the levels of inflammatory cytokines and molecules of MAPK pathway including IL-6, TNF-α, IL-1β, IL-22, p38 and phosphor-p38 expressions. Moreover, 16S rRNA sequencing of colitis murine fecal samples was applied to investigate the influence of supplementation of BT to the gut microbiota homeostasis. ResultsBoth live and heat-treated inactivated BT decreased the DAI and histological scores as well as levels of inflammatory factors, particularly IL-6 while increasing IL-22 of DSS-induced colitis murine models. The cell experiments showed that inactivated BT downregulates IL-6 expression in THP1 via inhibiting p38 phosphorylation and affecting M1 polarization. Moreover, the 16S rRNA sequencing results showed that BT and IBT gavage could increase beta-diversity of gut flora in DSS-induced colitis mice. Furthermore, the significance test for differences between the groups showed that BT could increase Faecalebaculum, Lactobacillus and Bacteroides, while decreasing Akkermansia. ConclusionIn summary, our findings imply that BT and its heat-treated inactivated bacteria exert a protective effect by suppressing macrophage-induced IL-6 through the inhibition of p38 MAPK pathway and ameliorating intestinal gut dysbiosis in experimental colitis.

Helicobacter pylori-induced fibroblast-derived Serpin E1 promotes gastric cancer growth and peritoneal dissemination through p38 MAPK/VEGFA-mediated angiogenesis

BackgroundFibroblasts, especially cancer-associated fibroblasts (CAFs), represent the predominant stromal cell population in the tumor microenvironment and have an important function in tumorigenesis by interacting with tumor cells. However, their interaction remains elusive in an inflammatory tumor microenvironment induced by Helicobacter pylori (H. pylori).MethodsThe expression of Serpin family E member 1 (Serpin E1) was measured in fibroblasts with or without H. pylori infection, and primary gastric cancer (GC) cells. Serpin E1 knockdown and overexpression fibroblasts were generated using Serpin E1 siRNA or lentivirus carrying Serpin E1. Co-culture models of fibroblasts and GC cells or human umbilical vein endothelial cells (HUVECs) were established with direct contact or the Transwell system. In vitro functional experiments and in vivo tumorigenesis assay were employed to study the malignant behaviors of GC cells interacting with fibroblasts. ELISA was used for quantifying the levels of Serpin E1 and VEGFA in the culture supernatant. The tube formation capacity of HUVECs was assessed using a tube formation assay. Recombinant human Serpin E1 (recSerpin E1), anti-Serpin E1 antibody, and a MAPK pathway inhibitor were utilized to treat HUVECs for elucidating the underlying molecular mechanisms.ResultsSerpin E1 was predominantly expressed in gastric CAFs. H. pylori infection significantly enhanced the expression and secretion of Serpin E1 by CAFs. Both fibroblast-derived Serpin E1 and recSerpin E1 enhanced the growth, invasion, and migration of GC cells, along with increased VEGFA expression and tube formation in HUVECs. Furthermore, the co-inoculation of GC cells and fibroblasts overexpressing Serpin E1 triggered the expression of Serpin E1 in cancer cells, which facilitated together xenograft tumor growth and peritoneal dissemination of GC cells in nude mice, with an increased expression of Ki67, Serpin E1, CD31 and/or VEGFA. These processes may be mediated by Serpin E1-induced migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs.ConclusionH. pylori infection induces Serpin E1 expression in fibroblasts, subsequently triggering its expression in GC cells through their interaction. Serpin E1 derived from these cells promotes the migration and p38 MAPK/VEGFA-mediated angiogenesis of HUVECs, thereby facilitating GC growth and peritoneal metastasis. Targeting Serpin E1 signaling is a potential therapy strategy for H. pylori-induced GC.

SHP2 inhibitors maintain TGFβ signalling through SMURF2 inhibition

Despite the promising antitumor activity of SHP2 inhibitors in RAS-dependent tumours, overall responses have been limited by their narrow therapeutic window. Like with all MAPK pathway inhibitors, this is likely the result of compensatory pathway activation mechanisms. However, the underlying mechanisms of resistance to SHP2 inhibition remain unknown. The E3 ligase SMURF2 limits TGFβ activity by ubiquitinating and targeting the TGFβ receptor for proteosome degradation. Using a functional RNAi screen targeting all known phosphatases, we identify that the tyrosine phosphatase SHP2 is a critical regulator of TGFβ activity. Specifically, SHP2 dephosphorylates two key residues on SMURF2, resulting in activation of the enzyme. Conversely, SHP2 depletion maintains SMURF2 in an inactive state, resulting in the maintenance of TGFβ activity. Furthermore, we demonstrate that depleting SHP2 has significant implications on TGFβ-mediated migration, senescence, and cell survival. These effects can be overcome through the use of TGFβ-targeted therapies. Consequently, our findings provide a rationale for combining SHP2 and TGFβ inhibitors to enhance tumour responses leading to improved patient outcomes.

Alpelisib decreases nevocytes of congenital melanocytic nevi.

Multiple, large or giant congenital melanocytic nevi (CMN) are uncommon and affected patients can show progressive growth and thickening, associate neurocutaneous melanocytosis or develop melanoma. Current treatment modalities are mostly complex surgeries that frequently do not solve the disease and its risks completely. Thus, investigation on new treatment options for CMN and its complications must continue. MAPK pathway inhibitors are being investigated, also targeting PI3K-AKT. Omipalisib (PI3K inhibitor, with no indications approved yet) has been studied for CMN invitro and in mice with promising results. However, alpelisib, a PI3K inhibitor approved with an adequate safety profile for patients with severe manifestations of PROS (PIK3CA-Related Overgrowth Spectrum), had not yet been tested for CMN. To evaluate the effect of alpelisib in nevocytes of congenital melanocytic nevi. Nevomelanocytic tissue samples of 10 patients were collected prospectively and, following a previously reported preclinical exvivo model, explants were placed in organotypic culture for 5 days, with or without alpelisib. Consecutively, tissue sections were stained and using scanned images with Qupath and ImageJ softwares, representative regions from the dermis were analysed (using Wilcoxon test and Spearman's correlation). When comparing alpelisib-treated explants with respect to control explants, we found a decrease in cell density (p = 0.0273), in density of SOX10+-cells (p = 0.0391) and also in the % of S-100+ area (p = 0.0078), in alpelisib samples. The three markers showed a positive correlation (p < 0.05). This study provides first-time evidence that alpelisib induces nevocyte reduction in CMN from patient-derived explants, probably inducted by autophagy. Alpelisib is an approved drug with an adequate safety profile used in another mosaicism affecting PI3K (PROS). Further studies are needed to evaluate its efficacy in treating CMN and potentially, their complications, either with local or systemic administration, alone or in combination.

Lycium barbarum polysaccharides attenuate oxidative stress and mitochondrial toxicity induced by mixed plasticizers in HepG2 cells through activation of Nrf2

AimsIn daily life, it is common for humans to be exposed to multiple phthalate esters (PAEs). However, there is limited research on the mechanisms and intervention of combined PAEs toxicity. This study aims to explore the cytotoxicity of combined PAEs and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. Main methodsLBP (62.5, 125 and 250 μg/mL) were applied to intervene HepG2 cells treated with DEHP and DBP mixtures (50, 100, 200, 400 and 800 μg/mL). Western Blot and different kits were mainly performed in our study. Key findingsDEHP and DBP mixtures suppressed the expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and activated MAPK pathway by increasing ROS. Combined DEHP and DBP exposure reduced ATP content and inhibited the mitochondrial biogenesis pathway in HepG2 cells through oxidative stress, which in turn caused cytotoxicity. LBP reduced oxidative stress and cell death induced by mixed plasticizers, upregulated Nrf2 levels and mitochondrial biogenesis pathway levels and inhibited MAPK pathway activation. Notably, after treating HepG2 cells with Nrf2-specific inhibitor (ML385, 0.5 μM), we found that the activation of Nrf2 played a crucial role on LBP intervention of DEHP and DBP induced HepG2 cytotoxicity. SignificanceThis study not only enhances our understanding of the toxicological effects caused by combined PAEs exposure, but also has significant implications in devising strategies to mitigate the toxicological consequences of combined exposure to exogenous chemicals through the investigation of the role of LBP.

Abstract A091: The class I selective, oral HDAC inhibitor bocodepsin enhances the response to MAPK pathway inhibitors in multiple tumor types with mutations in MAPK pathway signaling proteins

Abstract Activating MAPK pathway mutations, found in &amp;gt;30% of all human tumors, are frequently associated with poor outcomes. Several MAPK pathway targeted drugs are approved but single agent activity is modest, supporting the need for rational targeted combinations to improve outcomes. Multiple studies propose synthetic lethality between Class I HDAC inhibitors and MAPK pathway inhibitors in MAPK pathway mutated cancers, due to a combined effect of inhibiting double-stranded DNA repair and other survival pathways to drive apoptosis. The use of HDACi has been challenged by poor potency, selectivity, safety and convenience, highlighting the need for a better HDACi in the clinic. Bocodepsin, a potent class I-selective oral HDACi, is currently in a phase 1b/2 trial in combination with binimetinib in patients with NRAS mutant melanoma. The clinical profile shows exposure consistent with preclinical activity with strong pharmacodynamic activity at tolerated doses. OKI-005, a bocodepsin analog optimized for in vitro activity, produces the same active metabolite as bocodepsin. To explore the potential for broad application of bocodepsin combinations, we evaluated OKI-005 in combination with the MEK inhibitor binimetinib in a 72H in vitro proliferation screen of 89 cell lines covering a range of tumor types, MAPK pathway mutations, and co-mutations. Net cell killing with the combination vs. single agents was evaluated at clinically relevant doses. Combination activity was observed in tumor types that commonly harbor activating MAPK pathway mutations, including melanoma (6/13, 46%), pancreatic (3/5, 60%), lung (10/22, 45%) and colon (8/13, 62%). Among these, single agent binimetinib activity was observed in only 1/53. The combination response rate in colon cancer models was of note, as single agent MAPK pathway inhibitors are typically inactive in colon cancers. Activity was independent of the activating MAPK pathway mutation: combination activity was observed in lines with KRAS (13/24, 52%), NRAS (8/12, 67%) and BRAF (7/13, 54%) mutations. These findings translated to combinations with other MAPK inhibitors in vitro and in vivo. In vitro, OKI-005 exhibited enhanced activity in combination with KRAS inhibitors and with the ERK inhibitor GDC-0994. 25 of the 89 tumor lines were evaluated in combination with GDC-0994, and the profile of inhibition was identical to that with binimetinib. In vivo, the combination of binimetinib + bocodepsin led to 9/10 regressions in the NRAS mutant melanoma model SK-MEL-2, vs. 0/10 and 2/10 with binimetininb and bocodepsin, respectively. The combination of sotorasib + bocodepsin led to deeper regressions, vs. single agent sotorasib, in the KRAS mutant NSCLC model H358, and greater response in the KRAS mutant NSCLC model H2122. Combination activity was associated with increases in cleaved PARP and p-gH2AX, markers of DNA damage induction and apoptosis. These data demonstrate the potential for bocodepsin to synergize with MAPK pathway inhibitors across multiple indications in tumors that harbor MAPK pathway mutations. Citation Format: Rich Woessner, Maria Hoh, James Winkler, Tony Piscopio, Duncan Walker. The class I selective, oral HDAC inhibitor bocodepsin enhances the response to MAPK pathway inhibitors in multiple tumor types with mutations in MAPK pathway signaling proteins [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A091.

Abstract A165: FGFR alterations in pediatric cancers: Opportunity for targeted therapy

Abstract Introduction: The Fibroblast Growth Factor Receptor (FGFR) family consists of four members, FGFR1–4, and is involved in regulating cell proliferation, survival, and differentiation. FGFR alterations, such as fusions, mutations and amplification, have been found in multiple adult cancers, for which several FGFR inhibitors are in clinical development. Although cancer is a leading cause of death in children, the understanding of genetic causes of pediatric cancers is urgently lacking and the progress in developing targeted therapy in children significantly lags that in adults. This study aimed to uncover the FGFR alterations in pediatric cancers and provide preclinical rationale for FGFR inhibitors as potential therapy in children with cancer. Methods: The prevalence of FGFR alterations in pediatric cancers such as neuroblastoma (NB) , gliomas and sarcomas was calculated based on systematic queries of multiple cancer databases that house real-world molecular profiling data. A pan-FGFR inhibitor, BGJ398, was used as a tool compound for FGFR inhibition. Effect of BGJ398 on cell proliferation was tested by Incucyte in a panel of 12 NB cell lines. Cell migration, survival and signaling pathway modulation were carried out in a subset of NB lines. Results: The overall rates of genomic alterations (fusions, mutations and amplification) of FGFR1, 2, 3 and 4 in pediatric cancers are 0.5%, 0.1%, 1.1% and 0.8%, respectively, with pediatric gliomas exhibiting higher rates in all subtypes. Although FGFR genomic alterations are rare (&amp;lt;0.1%) in neuroblastoma and rhabdoid sarcoma, overexpression of FGFR1-4 is observed in subsets of these tumors. In pediatric glioma, FGFR1, 2, 3 and 4 alterations are found in 1%, 1.6%, 2.1% and 0.9% tumor samples, respectively, and the mutation profile substantially differs from that of adult glioma. Dose-dependent inhibition of cell proliferation and migration, as well as induction of cell death, were achieved with BGJ398 treatment in a panel of NB cells, accompanied by inhibition of MAPK pathway and induction of apoptosis. Conclusions: Adult and pediatric cancers share common mechanisms of FGFR activation, but differ in overall alteration rates and in relative abundance of specific aberrations. Preliminary preclinical data indicates the therapeutic potential of FGFR inhibitors in the treatment of certain pediatric cancers. Citation Format: Ivan Li, Yuchen Huo, Gary Li, Peter Zage. FGFR alterations in pediatric cancers: Opportunity for targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A165.

Abstract A099: Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors

Abstract KRAS is an oncogene implicated in a wide variety of tumors (~21% of solid tumors harbor KRAS mutations). Interaction of KRAS with its predominant Guanine Exchange Factor (GEF), SOS1, is crucial for activation of KRAS and further KRAS mediated oncogenic signaling. Thus, pharmacological inhibition of SOS1 is expected to be effective in treating RAS driven cancers. Biochemical potency of compounds was confirmed in a SOS1 mediated FRET based guanine exchange factor (GEF) assay. Multiple potent SOS1 inhibitors were identified with potencies in the low double digit nanomolar range. Selectivity was determined against SOS2 and a host of other kinases in the RAS/RAF/MEK or RAS/PI3K/AKT pathways. Anti-proliferative activity across a panel of WT and mutant KRAS cell lines revealed IC50 between 50-200 nM with synergism observed when combined with MAPK pathway inhibitors as well as the KRAS G12C inhibitor, Sotorasib, in a 3D anchorage independent assay format. Correspondingly, significant reduction in PD biomarkers (&amp;gt;50% at 100 nM), pERK and pAKT, was demonstrated in KRAS mutant as well as EGFR/PI3K mutant cell lines. PK-PD correlation was also established in a tumor bearing mice model, with dose-dependent reduction of both pERK and pAKT. Compounds demonstrated excellent ADME properties with significant tissue distribution upon oral dosing. One of the lead molecules demonstrated significant single agent efficacy with growth inhibition of approximately 70% in a KRAS G12C bearing NSCLC xenograft model. No adverse signs including reduction in food intake or body weight loss were reported during the study period. IND-enabling studies are currently ongoing for the lead compounds. Citation Format: Balakrishna Lakhavath, Manoj Pothuganti, Githavani Kummari, Hemasankar Pathange, Rajan Sekar, Srikant Viswanadha. Best in class, potent, SOS1 inhibitors demonstrate single agent activity in preclinical models of KRAS driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A099.

Abstract A101: Novel KRAS inhibitors suppress MAPK pathway signalling and display potent anti-proliferative activity across a broad range of KRAS mutant cell lines

Abstract KRAS is the most frequently mutated oncogene across different cancer types. Redx Pharma are developing novel, orally bioavailable, small molecule inhibitors of KRAS. These compounds display activity against a broad spectrum of the most frequently occurring KRAS mutations whilst maintaining selectivity over NRAS and HRAS protein isoforms. Isoform selective multi-KRAS inhibitors were identified using biochemical SOS1-independent nucleotide exchange assays (NEA) and validated by surface plasmon resonance (SPR) spectroscopy. Compounds of interest were the further triaged using relevant cancer cell lines for inhibition of phospho-ERK (pERK) and anti-proliferative effects both in 2 and 3-dimensional assay formats. KRAS selective inhibitors retaining activity across a spectrum of mutations were identified, these compounds exhibit picomolar affinity for KRAS, translating into low nanomolar antiproliferative activity. As further confirmation of MAPK pathway inhibition, treatment of relevant cancer cell lines with Redx compounds resulted in suppression of DUSP6 and SPTY4 gene expression. Cellular washout experiments indicated sustained inhibition of pERK post washout of compounds consistent with high affinity compounds with extended residency time at KRAS. Optimisation towards KRAS inhibitors suitable for clinical evaluation is ongoing. Citation Format: Inder Bhamra, Helen Mason, Simon Woodcock, Katie Anderson, James Kelly, Grace Haydon, Manuela La Montagna, Megan Brierley, Emma Bishop, Ross Keeler, James Ryan, Paula Jackson, Helen Aylott, Kelvin Ho, Daniel Leng, Clifford Jones, Caroline Phillips. Novel KRAS inhibitors suppress MAPK pathway signalling and display potent anti-proliferative activity across a broad range of KRAS mutant cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A101.

Abstract C026: Initial safety, pharmacokinetics, and recommended Phase 2 dose from RAMP 203: A Phase 1/2 study of Avutometinib + Sotorasib in KRAS G12C Mutant Non-Small Cell Lung Cancer

Abstract Background: KRAS mutations occur in ~30% of non-small cell lung cancer (NSCLC), KRAS G12C mutations being the most common (40%). KRAS G12C inhibitors (G12Ci) are FDA approved for KRAS G12C mutant (mt) NSCLC. Acquired mutations and amplifications occurring upon clinical progression may limit the benefit of G12Ci. Simultaneous targeting of multiple nodes in the RAS/MAPK pathway may improve response depth and durability. Avutometinib is a first-in-class oral RAF/MEK clamp that potently inhibits MEK kinase activity while also blocking the compensatory reactivation of MEK by upstream RAF. In preclinical G12Ci-naïve NSCLC models, addition of avutometinib improved depth of MAPK pathway inhibition and substantially enhanced tumor regression relative to sotorasib alone. Initial clinical efficacy of avutometinib in KRAS G12C NSCLC was observed in the FRAME study [NCT03875820]: 4/6 patients (pts) demonstrated tumor reduction including 1 PR. Methods: RAMP 203 is a phase 1/2, multicenter, open label, dose evaluation/expansion study evaluating the efficacy and safety of avutometinib + sotorasib in pts with KRAS G12C NSCLC [NCT05074810]. Pts with unresectable stage 3B-C and stage 4 KRAS G12C NSCLC and 1-3 prior systemic regimens were enrolled. Previous treatment with MEK inhibitors was excluded, but prior G12Ci treatment was allowed. Up to 3 dose levels (DL) of avutometinib (DL1, 3.2 mg PO BIW 21/28 days; DL2, 4.0 mg PO BIW 21/28 days; DL-1, 2.4 mg PO BIW 21/28 days) + sotorasib (960 mg PO QD 28/28 days) were evaluated in the Part A (dose evaluation, standard 3+3 design) to determine the Recommended Phase 2 Dose (RP2D, based on dose limiting toxicities [DLTs], other safety data, and pharmacokinetics) for Part B (dose expansion). Results: At the time of data cut, 10 pts (9 DLT evaluable) with KRAS G12C NSCLC (all stage 4) were enrolled to DL1 (n=3) and DL2 (n=7). Pts received a median of 3 prior systemic regimens (5 pts with previous G12Ci). No DLTs were observed in DL1 or DL2, leading to the selection of DL2 as RP2D. The majority of DL2 TRAEs observed were grades 1-2. The most common TRAEs of any grade with a frequency of ≥20% of pts included nausea, AAT increase, diarrhea, fatigue, and pruritus. The mean exposures (AUC) of avutometinib and sotorasib in DL1 and DL2 were within range of past single agent studies, indicative of minimal drug-drug interactions (DDIs). Preliminary efficacy data will be available at the time of presentation. Conclusions: The combinability of avutometinib and sotorasib was demonstrated in heavily-pretreated, advanced KRAS G12C NSCLC. Avutometinib 4.0 mg PO BIW 21/28 days + sotorasib PO 960 mg QD 28/28 days was established as the RP2D with no indications of DDIs. No new safety signals were observed and most TRAEs were mild to moderate. Enrollment in RAMP 203 Part B is ongoing. Citation Format: Mark M Awad, Jerome Goldschmidt, Alexander I Spira, Rajat Malhotra, Jeffrey T Yorio, Vijeta Bhambhani, Yaofeng Cheng, Pablo S Lee, Ramaswamy Govindan. Initial safety, pharmacokinetics, and recommended Phase 2 dose from RAMP 203: A Phase 1/2 study of Avutometinib + Sotorasib in KRAS G12C Mutant Non-Small Cell Lung Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C026.

Antioxidant effects of phenolic compounds in through the distillation of Lonicera japonica & Chenpi extract and anti-inflammation on skin keratinocyte

The phenolic compounds in Lonicera japonica & Chenpi distillation extract (LCDE) were thoroughly examined for their antioxidant and anti-inflammatory properties. Phenolic compounds in LCDE were analyzed for five peaks using high-performance liquid chromatography (HPLC) combined with mass spectrometry (MS) and determined. Five phenolic compounds were identified from the samples and MS data. Ultrafiltration with LC analysis was used to investigate the ability of bioactive compounds to target DPPH. As a result, it was confirmed that the major compounds exhibited a high binding affinity to DPPH and could be regarded as antioxidant-active compounds. In addition, the anti-inflammatory effect of LCDE was confirmed in vitro, and signal inhibition of anti-inflammation cytokines, MAPK and NF-kB pathways was confirmed. Finally, Molecular docking analysis supplements the anti-inflammatory effect through the binding affinity of selected compounds and inflammatory factors. In conclusion, the phenolic compounds of the LCDE were identified and potential active compounds for antioxidant and anti-inflammatory activities were identified. Additionally, this study will be utilized to provide basic information for the application of LCDE in the pharmaceutical and pharmaceutical cosmetics industries along with information on efficient screening techniques for other medicinal plants.

PATH-54. CDC42BPA::BRAF REPRESENTS A NOVEL FUSION IN DESMOPLASTIC INFANTILE GLIOMA

Abstract Desmoplastic infantile ganglioglioma/desmoplastic infantile astrocytomas (DIG/DIA), although rare, are low-grade glioneuronal tumors that typically affect patients &amp;lt; 1 year old, and generally carry a good prognosis. Often successfully cured with surgical excision, DIG/DIA still carry the potential for malignant transformation or recurrence. Whilst pediatric low grade glioma (LGG) survival rates are &amp;gt;95%, tumor grade does not correlate well with outcomes in infant LGG (iLGG), thus highlighting the need for investigation into molecular mechanisms of tumor progression. We describe a novel CDC42BPA-BRAF fusion in a DIA in a three-month old patient, and its detection via Next Generation Sequencing. Future studies will investigate whether this novel fusion can function as a target for BRAF and MAPK pathway inhibitors, which have recently been FDA-approved for pLGG.

DDDR-11. IDENTIFYING NEW VULNERABILITIES TO TARGET PEDIATRIC LOW-GRADE GLIOMA

Abstract Pediatric low-grade gliomas (pLGGs) are the most common solid tumors in children and are associated with devastating lifelong morbidities and mortality. Recent genomic profiling efforts have revealed that these tumors are largely driven by single-driver events that activate MAPK signaling. These insights have led to early clinical trials evaluating the role of MAPK inhibitors for these children with promising initial results. However, pLGGs are not cured by MAPK inhibitors, and tumors often rapidly rebound upon cessation of treatment. Therefore, continuous dosing of these inhibitors is required throughout a child’s development–often with significant and potentially permanent toxicities. In addition, 30-40% of tumors do not respond to MAPK inhibition, indicating primary resistance. We hypothesized that pediatrlc low-grade gliomas may also harbor additional dependencies beyond the MAPK pathway that may represent potential therapeutic targets. To address this, we generated isogenic neural stem cell models that expressed pLGG relevant oncogenes (BRAF family members, FGFR family members and MYB transcription factors, and subjected them to genome-scale CRISPR-cas9 screens. Compared to vector controls, pLGG models exhibited genetic dependencies on pathways that included regulators of cell-cycle and differentiation, in addition to genes involved in regulating the mTOR and MAPK pathways. Cell lines expressing MYB family transcription factors also harbored dependencies on genes involved in the DREAM complex. Together, these genes and pathways represent potential targets for combination treatments with MAPK pathway inhibitors for the treatment of pediatric low-grade gliomas.

Responses to Combination Chemotherapy with MAPK Pathway Inhibition in Relapsed and Refractory LCH

MAPK pathway inhibition (MAPKi) is associated with high response rates in patients with Langerhans cell histiocytosis (LCH), but monotherapy may not be curative and efficacy in LCH-associated neurodegeneration (LCH-ND) is not known. Based on upregulation of Bcl-xL by MAPK activation, we hypothesize that MAPKi may render LCH cells more sensitive to chemotherapy-induced cytotoxicity. We assessed the safety and efficacy of combination therapy in 10 patients with refractory systemic disease and LCH-ND. Records of patients treated with combination therapy at Texas Children's Hospital from October 2018 to July 2023 were reviewed for clinical responses as well as peripheral blood/tissue BRAFV600E mutation status, ataxia rating scores, and toxicity. The median age at the start of combination therapy was 6.82 years (range, 0.48-20.45 years) with a median of 4 treatments prior to this therapy regimen (range, 1-14). Almost all Clofarabine was administered outpatient (typical starting dose 25 mg/m2/day x 5 days). MAPK inhibitors included BRAFV600E inhibitors (50%) and MEK inhibitors (50%). Overall response was 8/10 (80%), with 5/5 (100%) objective response rate (ORR) for systemic disease. Further, 5/6 (83%) demonstrated reduced or extinguished peripheral BRAFV600E+ fraction by the end of combination therapy. Ataxia rating scores were improved in 2/3 (67%) individuals with clinical neurodegeneration. Common toxicities during treatment included fever, skin rash, arthralgia, and cytopenia. Combining therapy was well-tolerated and associated with clinical and molecular improvement in most patients with highly refractory systemic LCH and LCH-ND. Further prospective multi-center trials are required to optimize this approach, determine potential for combination therapy to achieve cure, and compare risks and benefits to chemotherapy or MAPK inhibitor monotherapy.

Threshold Attenuation of Perk Signaling Is Sufficient to Overcome RAS-Mediated FLT3 Inhibitor Resistance

Introduction Acute myeloid leukemia (AML) with activation of FMS-related receptor tyrosine kinase 3 ( FLT3) confers a poor prognosis, principally due to disease relapse. The FLT3 inhibitor (FLT3i) gilteritinib is standard of care in relapsed/refractory FLT3-mutant AML, however its efficacy is limited by drug resistance, primarily due to RAS mutations. This suggests that RAS signaling is the critical mediator of downstream FLT3 signaling, but there is no standardized treatment for patients who relapse on FLT3i due to RAS pathway activation. Efforts to overcome resistance have been limited both by understanding of the optimal RAS effectors to target as well as the potential toxicity of simultaneously targeting FLT3 and RAS. Here, we demonstrate that while RAS-driven resistance to gilteritinib is mediated through MAPK, not PI3K, attenuation of pERK signaling is sufficient to overcome this resistance both in vitro and in vivo. Methods/ Results FLT3-ITD+ cells (Molm14) with transduced NRAS mutations are resensitized to gilteritinib by MEK inhibitor treatment, proving that MAPK signaling is necessary for RAS-driven resistance. Further, expression of a constitutively active MEK construct (MEKDD) in Molm14 confers resistance to gilteritinib, showing MAPK signaling is sufficient for RAS-driven resistance. Conversely, we show that activated PI3K signaling is neither necessary nor sufficient to drive resistance, as AKT inhibition cannot resensitize NRAS-mutants to gilteritinib, and an activated AKT mutant does not confer gilteritinib resistance. We used a doxycycline-inducible system to increase expression of MEKDD, to titrate levels of ERK signaling activation in Molm14 cells. Surprisingly, we found that the relationship between ERK phosphorylation (pERK) and FLT3i resistance was non-linear and appeared to increase at a threshold of pERK (62%). In keeping with this, suppression of pERK levels in Molm14 cells with concurrent RAS mutations to levels below 70% of baseline using the MEK inhibitor (MEKi) trametinib, was sufficient to resensitize to gilteritinib in proliferation assays. A similar phenomenon was seen in apoptosis assays. Molm14 NRAS Q61K mutant cells treated with 500nM gilteritinib plus increasing doses of trametinib showed apoptosis plateaued at the relatively low dose of 5nM trametinib. Western Blot analysis showed residual pERK activation at this dose. Thus, attenuation of MAPK signaling below a threshold, rather than its ablation, can overcome FLT3i resistance. In a patient-derived xenograft (PDX) model of AML with concurrent FLT3-ITD and NRAS Q61K, the combination of low dose MEK inhibition (PD-901 1.5mg/kg) combined with gilteritinib was equivalent to standard dose MEK inhibition (PD-901 5mg/kg) combined with gilteritinib in suppressing outgrowth of human CD45+ cells in spleen, bone marrow, and blood of NSGS mice in comparison to vehicle-treated control. Standard dose MEK inhibition in combination with gilteritinib was also noted to be intolerable in the majority of mice with only 2 of 6 mice surviving 28 days. Given the similar activity of low and high dose MAPK inhibition in combination with gilteritinib, we hypothesized that suppression of ERK activation below a critical level is sufficient to activate a shared transcriptional program to trigger cell cycle arrest and apoptosis. To test this hypothesis, we performed RNAseq analysis of Molm14 NRAS Q61K cells treated with gilteritinib, standard dosing of trametinib, low dose trametinib, or the combination. There is a gene expression signature of MAPK expression unique to the combination of gilteritinib and trametinib that is dose independent of trametinib, which includes MAPK regulators including ETV4 and ETV5. Conclusions MAPK is the critical effector pathway of RAS-driven gilteritinib resistance, but partial attenuation of pERK signaling is sufficient to inhibit proliferation and induce apoptosis, restoring sensitivity to FLT3i. Applied clinically, this suggests feasibility of low dose MAPK inhibition in combination with gilteritinib to treat resistant FLT3-activated AML. This strategy that would improve tolerability, a major concern in clinical use of MAPK pathway inhibitors.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS–MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

Silkworm pupa protein peptide improved DSS-induced colitis in C57BL/6 mice through the MAPK/NF-κB signaling pathway

Numerous studies have revealed the multifunctional activities of silkworm pupa peptides (SPP). In this study, we investigated the protective effects of SPP on dextran sulfate sodium (DSS)-induced colitis in mice. The results showed that SPP improved the clinical symptoms of colitis in mice, including increased colon length, slowed weight loss, and reduced disease activity index (DAI) scores. Furthermore, SPP significantly alleviated DSS-induced colonic injury by reducing the expression of myeloperoxidase (MPO) in colonic tissues, enhancing mucin secretion in the colon, and decreasing colonic cell apoptosis. SPP also mitigated the severity of colitis by modulating serum inflammatory factors and improving colonic oxidative stress levels. Western blot analysis revealed that SPP suppressed the activation of the MAPK and NF-κB signaling pathways. In conclusion, SPP protected against DSS-induced colitis in mice by reducing cell apoptosis, attenuating inflammatory responses, and improving oxidative stress levels, potentially through the inhibition of MAPK and NF-κB signaling pathways.

Peroxisome disruption alters lipid metabolism and potentiates antitumor response with MAPK-targeted therapy in melanoma.

Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK pathway inhibitors (MAPKis) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the proapoptotic effects of MAPKis via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Cotargeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKis and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy consisting of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor, and MAPKis demonstrated potent antitumor activity in preclinical melanoma models, thus representing a promising approach for melanoma treatment.

Total Flavonoids from Camellia oleifera Alleviated Mycoplasma pneumoniae-Induced Lung Injury via Inhibition of the TLR2-Mediated NF-κB and MAPK Pathways.

Mycoplasma pneumoniae (M. pneumoniae) is an atypical bacterial pathogen responsible for community-acquired pneumonia primarily among school-aged children and young adults. Camellia oleifera (C. oleifera) has been used as a medicinal and edible plant in China for centuries, the constituents from which possessed various bioactivities. Notably, flavonoids existing in residues of C. oleifera defatted seeds exhibited significant anti-inflammatory activities. In the present study, we investigated the impact of total flavonoids from C. oleifera (TFCO) seed extract on M. pneumoniae pneumonia. TFCO was obtained using multiple column chromatography methods and identified as kaempferol glycosides via UPLC-HRESIMS. In a M. pneumoniae pneumonia mouse model, TFCO significantly reduced the lung damage, suppressed IL-1β, IL-6, and TNF-α production, and curbed TLR2 activation triggered by M. pneumoniae. Similarly, in RAW264.7 macrophage cells stimulated by lipid-associated membrane proteins (LAMPs), TFCO suppressed the generation of proinflammatory cytokines and TLR2 expression. Moreover, TFCO diminished the phosphorylation of IκBα, JNK, ERK, p38, and p65 nuclear translocation in vitro. In conclusion, TFCO alleviated M. pneumoniae-induced lung damage via inhibition of TLR2-mediated NF-κB and MAPK pathways, suggesting its potential therapeutic application in M. pneumoniae-triggered lung inflammation.